Figure 2.
Endothelial cells (ECs) bind sCD177/PR3 specifically via PECAM-1. (A) HUVECs were pretreated with TNF-α overnight and incubated with saline, rCD177, rPR3, or sCD177/PR3 (2 µg/mL) for 4 hours at 37°C. HUVECs were analyzed by flow cytometry using direct fluorescence-labeled mAbs against CD177 (MEM166) or PR3 (PR3D1). Values are presented as mean fluorescence intensity (MFI) ± standard deviation (SD) (n = 5). (B) TNF-α–pretreated HUVECs were treated with mAbs (1 µg/mL), against PECAM-1 domain 1 (Gi18) or against PECAM domains 2 and 6 (PECAM 1.1 or PECAM 1.2, respectively), and subsequently incubated with sCD177/PR3 (2 µg/mL) for 4 hours at 37°C. HUVECs were analyzed by flow cytometry using direct fluorescence-labeled anti-CD177 (MEM166). Values are presented as MFI ± SD (n = 5). (C) Presence of CD177/PR3 and ECs after incubation with granulocytes. Neutrophils were isolated from whole blood of CD177-positive and CD177-negative phenotyped donors. HUVECs were incubated with granulocytes for 4 hours in 37°C. Presence of CD177 or PR3 on ECs was determined by flow cytometry. Bar graphs represent the MFI on the EC membrane. Values are presented as mean ± SD (n = 5). (D) Presence of CD177/PR3 on neutrophils after incubation with serum. CD177-negative and CD177-positive phenotyped neutrophils were isolated and incubated for 2 hours with 1 mL of serum from CD177-positive donors. Presence of CD177, PR3, and PECAM-1 on the neutrophil surface before and after incubation was determined by flow cytometry. Bar graphs represent the MFI on the neutrophil surface. Values are presented as mean ± SD (n = 5). (E) Binding of rCD177, rPR3, and sCD177/PR3 on coated PECAM-1 was determined in real-time by using SPR. Purified PECAM-1 was immobilized on a sensor chip. mAbs against PECAM-1 Ig domains 5 + 6 or 1 + 2 or rCD177, rPR3, or sCD177/PR3 were injected at 25°C. Note that rCD177 does not bind to PECAM-1 (upper panel). To calculate the KD for PR3 or sCD177/PR3 interaction with PECAM-1, different concentrations of both proteins were run as analyte on coated PECAM-1 (middle and lower panel). (F) ECs express FCGRI and FCGRII on the surface. The expression of FCGRs (I, II, and IIIb) on HPMEC monolayer were evaluated in ELISA. Endothelial monolayers were cultured in a 96-well plate and blocked with bovine serum albumin. mAbs against FCGRs (10.1 for FCGRI, AT10 for FCGRII, and 3G8 for FCGRIII) were added to ECs, and bound antibodies were detected by using horseradish peroxidase–labeled anti-mouse secondary antibodies. The optical densities (ODs) were evaluated in photometer.**P< .01, n.s., not significant.

Endothelial cells (ECs) bind sCD177/PR3 specifically via PECAM-1. (A) HUVECs were pretreated with TNF-α overnight and incubated with saline, rCD177, rPR3, or sCD177/PR3 (2 µg/mL) for 4 hours at 37°C. HUVECs were analyzed by flow cytometry using direct fluorescence-labeled mAbs against CD177 (MEM166) or PR3 (PR3D1). Values are presented as mean fluorescence intensity (MFI) ± standard deviation (SD) (n = 5). (B) TNF-α–pretreated HUVECs were treated with mAbs (1 µg/mL), against PECAM-1 domain 1 (Gi18) or against PECAM domains 2 and 6 (PECAM 1.1 or PECAM 1.2, respectively), and subsequently incubated with sCD177/PR3 (2 µg/mL) for 4 hours at 37°C. HUVECs were analyzed by flow cytometry using direct fluorescence-labeled anti-CD177 (MEM166). Values are presented as MFI ± SD (n = 5). (C) Presence of CD177/PR3 and ECs after incubation with granulocytes. Neutrophils were isolated from whole blood of CD177-positive and CD177-negative phenotyped donors. HUVECs were incubated with granulocytes for 4 hours in 37°C. Presence of CD177 or PR3 on ECs was determined by flow cytometry. Bar graphs represent the MFI on the EC membrane. Values are presented as mean ± SD (n = 5). (D) Presence of CD177/PR3 on neutrophils after incubation with serum. CD177-negative and CD177-positive phenotyped neutrophils were isolated and incubated for 2 hours with 1 mL of serum from CD177-positive donors. Presence of CD177, PR3, and PECAM-1 on the neutrophil surface before and after incubation was determined by flow cytometry. Bar graphs represent the MFI on the neutrophil surface. Values are presented as mean ± SD (n = 5). (E) Binding of rCD177, rPR3, and sCD177/PR3 on coated PECAM-1 was determined in real-time by using SPR. Purified PECAM-1 was immobilized on a sensor chip. mAbs against PECAM-1 Ig domains 5 + 6 or 1 + 2 or rCD177, rPR3, or sCD177/PR3 were injected at 25°C. Note that rCD177 does not bind to PECAM-1 (upper panel). To calculate the KD for PR3 or sCD177/PR3 interaction with PECAM-1, different concentrations of both proteins were run as analyte on coated PECAM-1 (middle and lower panel). (F) ECs express FCGRI and FCGRII on the surface. The expression of FCGRs (I, II, and IIIb) on HPMEC monolayer were evaluated in ELISA. Endothelial monolayers were cultured in a 96-well plate and blocked with bovine serum albumin. mAbs against FCGRs (10.1 for FCGRI, AT10 for FCGRII, and 3G8 for FCGRIII) were added to ECs, and bound antibodies were detected by using horseradish peroxidase–labeled anti-mouse secondary antibodies. The optical densities (ODs) were evaluated in photometer.**P< .01, n.s., not significant.

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